Nucleic acid amplification may be used to determine whether a particular template nucleic acid is present in a sample. If an amplification product is produced, this indicates that the template nucleic acid was present in the sample. Conversely, the absence of any amplification product indicates the absence of template nucleic acid in the sample. Such techniques are of great importance in diagnostic applications, for example, for determining whether a pathogen is present in a sample.
Nucleic acids may be amplified by a variety of thermocycling and isothermal techniques. Thermocycling techniques, such as the polymerase chain reaction (PCR), use temperature cycling to drive repeated cycles of DNA synthesis leading to large amounts of new DNA being synthesised in proportion to the original amount of template DNA. Recently, a number of isothermal techniques have also been developed that do not rely on thermocycling to drive the amplification reaction. Isothermal techniques which utilise DNA polymerases with strand-displacement activity have been developed for amplification reactions that do not involve an RNA-synthesis step. Similarly, for amplification reactions that do involve an RNA-synthesis step, isothermal techniques have been developed that use reverse transcriptase, RNase H and a DNA-dependent RNA polymerase.
The products of nucleic acid amplification reactions have traditionally been analysed using gel electrophoresis (either agarose or acrylamide-based) using a fluorescent dye (such as ethidium bromide) to stain for the presence of DNA. This method can be used to indicate the number, amount and size of the amplified products. However, the preparation, running and analysis of amplification reactions using gel electrophoresis requires extensive manual intervention and hazardous reagents and is time-consuming (typically taking around 1 hour in total). In addition, multiple PCR cycles (typically 30) are required to produce detectable product. More recently, methods with increased sensitivity over gel electrophoresis have been developed which rely on fluorescence-based techniques or a turbidity assay to monitor the products of nucleic acid amplification reactions in real-time.
A characteristic of DNA and RNA polymerases is the fact that they release the compound pyrophosphate (PPi) each time they incorporate a new base into the growing DNA/RNA molecule. Thus PPi is produced as a side product in a stoichiometric amount as nucleotides are added to a growing nucleotide chain by the polymerase. Thus it follows that the concentration of PPi is proportional to the amount of nucleic acid synthesis that has occurred and therefore to the accumulation of amplicon. For a polymer of length n, the reaction may be shown as:

A sensitive assay for PPi is known as the Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay (ELIDA) (see Nyren, P. and Lundin, A., Anal. Biochem. 151: (2) 504-509 (1985)). This assay has two steps: (1) conversion of pyrophosphate (PPi) to ATP by the enzyme ATP sulphurylase, and (2) utilisation of the ATP to produce light in the presence of luciferin and oxygen, catalysed by luciferase:

The use of ELIDA-type assays is advantageous in that bioluminescence readings can be rapidly obtained from small sample volumes and the readings can be made using simple, cheap monitoring devices such as photographic film or charge-coupled device (CCD) cameras.
U.S. Pat. Nos. 5,534,424, 5,498,523, WO 98/28440, WO 98/13523 and WO 02/20836 describe the use of ELIDA-based methods for sequencing short regions of DNA. The ELIDA assay was used to follow the incorporation of single nucleotides into a DNA molecule by a polymerase during a single round of polymerisation during pyrosequencing. Pyrosequencing is an iterative technique whereby only one of the four deoxynucleotide triphosphates (“dNTPs”) is present in each of the iterative assays to enable each deoxynucleotide triphosphate (“dNTP”) to be tested at each position of the sequence. Thus all of the components necessary for DNA synthesis are never present simultaneously.
The use of an end-point ELIDA-type assay termed ‘H3PIM’ for monitoring a thermocycling polymerase chain reaction (“PCR”) has also been described (see WO 92/16654 and Tarbary et al., J. Immunological Methods, 156 (1992) 55-60). Aliquots of the reaction mixture were taken at predetermined regular time intervals throughout the reaction process and/or at the end of the amplification process. Thus a lengthy stepwise assay involving the multiple addition of reagents is described.
WO 02/064830 describes the use of an ELIDA assay to perform an end-point assay for monitoring a thermocycling PCR reaction. In WO 02/064830 the ELIDA assay can be performed in a single step, whereas in WO 92/16654 multiple additions and an incubation step are required for monitoring thermocycling PCR as an end-point assay.
There are a number of problems associated with the end-point assays described above. Firstly, they require the components of the bioluminescence assay to be added to the reaction mixture following the amplification reaction. Opening of the tube may lead to contamination of the sample and moreover, to contamination of the laboratory. If the sample itself becomes contaminated then this could result in false-positives or false-negatives being generated. Moreover, if the laboratory becomes contaminated with the amplified template nucleic acid, this increases the likelihood that future samples will become contaminated and false-positive results or false-negative results will be obtained (for example, see Victor, T. et al., ‘Laboratory experience and guidelines for avoiding false-positive polymerase chain-reactions results’, Eur. J. Clin. Chem. & Clin. Biochem., 31(8): 531-535 (1993)). Thus the possibility of contamination represents a severe disadvantage in the use of end-point analysis of this type in diagnostic methods.
A further problem with the use of end-point analysis as described above is that dATP also acts as a substrate for luciferase. Thus when dATP is used as a substrate for the polymerase, spectral interference results from dATP instead of ATP reacting with the luciferase. WO 02/064830 describes how when dATP is used as the substrate in the amplification reaction, the light signal from the ELIDA rapidly decays. This decay would be a serious obstacle to the utility of an endpoint assay as the light reading measured would not only be a function of PPi concentration but also of time. Hence, if the endpoint assays are not performed with strict timing, they will not be quantitative.
An alternative to end-point assays are assays which are able to monitor the synthesis of nucleic acid during an amplification reaction in ‘real-time’, i.e., as the nucleic acid synthesis is progressing. Existing real-time assays include fluorescence-based techniques and turbidity assays.
Fluorescence-based techniques work by monitoring the change in fluorescence that is associated with the accumulation of an amplification product by some means. For example, methods for monitoring the amplification of DNA during PCR using double-stranded DNA-binding dyes (specifically hybridisation probes containing donor and acceptor fluorophores) are described in U.S. Pat. No. 5,994,056, WO 97/44486, WO 99/42611 and U.S. Pat. No. 6,174,670. These real-time fluorescence-based techniques make it possible to follow PCR without liquid sampling, thus avoiding the need for the reaction tube to be opened and therefore decreasing the risks of contamination.
However, fluorescence-based techniques have significant drawbacks. In particular, the cost of fluorescent reagents, particularly fluorescently-labelled primers, is high and sample preparation can be cumbersome. Further, the application of fluorescence-based systems may be hampered by the limited capacity of equipment and its high cost. Normally, a computer-driven integrated thermocycler-fluorimeter is required as the methods often follow PCR in real-time rather than being employed for end-point analyses. As a result, the accessibility (cost), and portability of such systems is compromised. Since detection is carried out within the PCR instrument, such methods are only available to suitably equipped laboratories.
Real-time turbidity assays involve monitoring the presence or absence of a white precipitate of magnesium pyrophosphate in the amplification reaction mixture as a method of determining whether PPi has been produced. This has been described as a method for determining whether or not an isothermal loop-mediated amplification reaction has occurred (see Mori, Y. et al., ‘Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation’, Biochem. and Biophys. Res. Comm., 289, 150-154 (2001)). However, this method is not very sensitive and requires PPi concentrations of around 0.6 mM before significant turbidity is observed. Further, the turbidity method also requires the use of absorbance as the means of measurement, thus complicating the hardware required to perform the assay.
Methods for bioluminometric quantification of PCR products are described in WO 01/42496 and Nygren et al., (‘Quantification of HIV-1 using multiple quantitative polymerase chain reaction standards and bioluminometric detection’, Analytical Biochem., 288, 28-38 (2001)). The products obtained as a result of PCR amplification were immobilised and quantified by means of strand extension polymerisation, in which one round of polymerisation was carried out at room temperature. The PPi released due to nucleotide incorporation was detected by an ELIDA-type system involving ATP sulphurylase and adenosine 5′-phosphosulphate (APS), using a luminometer and a potentiometric recorder to measure the signal one minute after the reaction was started.